This is a proposal for renewal of our grant supporting work on imitating and understanding enzymic catalysis through the study of catalytic antibodies. Catalytic antibody technology represents one of the most versatile strategies for creating new enzymes to emerge in the last several years: antibodies raised against transition state analogs have catalyzed more than 50 chemical transformations with significant rate accelerations and high selectivity. The immediate challenge in this field is to generate catalysts with even greater chemical efficiency. The specific aims of this proposal address this issue in three ways. First, the structure and mechanism of four representative antibody catalysts will be investigated in detail. These agents catalyze a Claisen rearrangement, a bimolecular Diels-Alder cycloaddition, a medium-sensitive decarboxylation, and a base-dependent elimination reaction, respectively. Their study will contribute to our understanding of the basic principles of catalysis, including the roles of transition state complementarity, proximity effects, desolvation, and chemical catalysis, providing information relevant to the creation of more active catalysts. Second, by refining transition state analog design and by developing and implementing more effective screening technologies we hope to optimize the strategies that have been developed to generate individual catalytic antibodies. Third, genetic selection will be investigated as a tool for augmenting the activity of first-generation catalytic antibodies. By characterizing the genetic changes that accumulate as the antibody becomes more active we may gain fundamental insight into the evolution of molecular function. We anticipate that the lessons learned in this project will lead to the elaboration of general strategies for designing highly active artificial enzymes of wide applicability in chemistry, biology, and medicine. Specifically, we hope to create practical antibody catalysts for the synthesis of vitamin B6 and for the hydrolysis of phosphate monoesters.